This invention relates to probes for, and methods of, detecting and measuring enzyme-mediated digestion of fibrinogen. More particularly, the invention relates to an antibody specifically reactive with a domain of fibrinogen and with a metabolic product of fibrinogen degradation, and a method of use of the antibody.
The clotting of blood is part of the body's natural response to injury or trauma, and is crucial to stopping loss of blood in such situations. Blood clot formation derives from a series of events called the coagulation cascade, the final steps of which involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen into fibrin, a mesh-like structure that forms the insoluble framework of the blood clot. The hemostasis enabled by clot formation is often a life-saving process in response to trauma, serving to arrest the flow of blood from severed vasculature.
The life-saving process of clot generation in response to an injury can, however, become life-threatening when it occurs at inappropriate places in the body or at inopportune moments. For example, a clot can obstruct a blood vessel and thereby reduce or stop the supply of blood to an organ or other body part. In addition, the deposition of fibrin contributes to partial or complete stenosis of blood vessels, resulting in chronic diminution of blood flow. Equally life-threatening are clots that become detached from their original sites and flow through the circulatory system causing blockages at remote sites. Such clots are known as embolisms. Indeed, pathologies of blood coagulation, such as heart attacks, strokes, and the like, have been estimated to account for approximately fifty percent of all hospital deaths.
Fibrinogen is one of the more well-studied and abundant proteins in the human circulatory system. By the late 1960s, the general subunit structure of fibrinogen was firmly established (Blomback 1968) and, a decade later, the complete amino acid sequence was reported (Lottspeich et al. 1977; Henschen et al. 1977; Henschen et al. 1979; Doolittle et al. 1979). Over the next 10 years, the cluster of three separate genes encoding the .alpha. (alpha), .beta. (beta), and .gamma. (gamma) subunits was identified on chromosome 4q23-q32 (Kant et al. 1985), and the apparently complete genetic sequences of all three fibrinogen subunits were published (Chung et al. 1991).
Fibrinogen (also abbreviated herein as "Fg") is a heavily disulfide-bonded homodimeric protein, composed of two symmetrical units (monomers), each including single copies each or three polypeptide chains: the A.alpha. (alpha), B.beta. (beta), and .gamma. (gamma) chains. Thus, fibrinogen has the generic structure (A.alpha.B.beta..gamma.).sub.2. For a review see Doolittle (1987). All three of the fibrinogen subunits have coiled domains, which permit the subunits to engage one another to form a "coiled coil" region in the fibrinogen monomer. In addition, the B.beta. and .gamma. chains each have a globular domain, while the A.alpha. chain is present in two forms; a predominant form having no corresponding globular domain (A.alpha.), and a less prevalent form in which a globular domain is present (A.alpha..sub.E) (Fu et al. 1992; 1994). Accordingly, because fibrinogen is homodimeric and because two forms of the A.alpha. subunit have been identified, two principal forms of fibrinogen are recognized: (A.alpha.B.beta..gamma.).sub.2 and (A.alpha..sub.E B.beta..gamma.).sub.2.
Fibrinogen's complex structure, and its central role in blood clot formation and wound healing account for the high profile it has enjoyed as a subject of both biochemical and medical research. Recently, new attention has been given to structure/function relationships in the fibrinogen molecule. This new interest has in part been prompted by growth in the understanding of this protein's range of activity in normal and pathological states (Blomback 1991; Bini et al. 1992; Dvorak 1992). Moreover, antibodies have been developed that are specifically reactive with or specifically bind to only some of the fragments, thereby permitting molecular identification of certain fragments with great accuracy and precision (Kudryk et al. 1989a). While a monoclonal antibody with specificity for human fibrinopeptide A has been identified (Kudryk et al. 1989b; see also European Patent Specification EP 0 345 811 B1), no comparable probe specific for fibrinopeptide B has yet been reported. However, despite these advances, the complexity of fibrinogen and its metabolic system have to date eluded complete elucidation.
Fibrinogen is synthesized and secreted into the circulation by the liver. Circulating fibrinogen is polymerized under attack by thrombin to form fibrin, which is the major component of blood clots or thrombi. Subsequently, fibrin is depolymerized under attack by plasmin to restore the fluidity of the plasma. Many of the steps in the polymerization and depolymerization processes have been well established (Doolittle 1984). The elevated levels of fibrinogen that are part of the acute phase response occurring in the wake of infections and trauma are now known to come from increased hepatic production, primarily in response to interleukin-6 (IL-6) (Sehgal et al. 1989).
The degradation of fibrinogen through hydrolytic cleavage by thrombin yields two forms of fibrin. The first form, "fibrin I," is defined by thrombin-mediated cleavage of the .alpha. chain (or the "A.alpha." chain) at A.alpha.16-17. This cleavage releases two short N-terminal peptides designated "fibrinopeptide A" or "FPA" (A.alpha.1-16). Subsequently, thrombin cleaves the .beta. chain (or the "B.beta." chain) at B.beta.14-15 to create "fibrin II." The cleavage of the B.beta. chain at B.beta.14-15 releases a peptide known as "fibrinopeptide B" or "FPB" consisting of the first 14 amino acid residues of the B.beta. chain (B.beta.1-14). See Blomback (1991). It is only after the thrombin cleavage of fibrinogen that the polymerization of fibrin occurs. Fibrinopeptide B, especially, is closely related to onset of fibrin polymerization.
In wound repair, fibrinogen serves as a key protein, achieving rapid arrest of bleeding following vessel injury. It promotes both the aggregation of activated platelets with one another to form a hemostatic plug, as well as endothelial cell binding at the site of injury to seal the margins of the wound. As the most abundant adhesive protein in the blood, fibrinogen attaches specifically to platelets, endothelial cells and neutrophils via different integrins (Hynes 1992). Five putative receptor recognition domains on human fibrinogen, distributed over its three subunits, have been identified by in vitro and in vivo analyses (Kloczewiak et al. 1984; Cheresh et al. 1989; Loike et al. 1991; Farrell et al. 1992; Gonda et al. 1982; Ribes et al. 1989).
Elevated levels of fibrinogen have been found in patients suffering from clinically overt coronary heart disease, stroke and peripheral vascular disease. Although the underlying mechanisms remain speculative, recent epidemiological studies leave little doubt that plasma fibrinogen levels are an independent cardiovascular risk factor possessing predictive power that is at least as high as that of other accepted risk factors such as smoking, hypertension, hyperlipoproteinemia or diabetes (Ernst 1990; Ernst et al. 1993). The structure of fibrin has been analyzed extensively in vitro (Doolittle 1984). Only recently, however, has attention been paid to the molecular structure of human thrombi and atherosclerotic plaques with respect to fibrinogen and fibrin products (Bini et al. 1987). Whereas thrombi formed in vivo consist primarily of fibrin II cross-linked by factor XIIIa, fibrinogen itself is a major component of uncomplicated atherosclerotic lesions, particularly fibrous and fatty plaques. Immunohistochemical as well as immunoelectrophoretic analyses indicate that fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue transglutaminase without becoming converted to fibrin (Valenzuela et al. 1992). Further understanding of these issues awaits the development of methods for the differential determination of fibrinogen subtypes in medical samples.
Fibrinogen-derived protein is also a major component of the stroma in which tumor cells are embedded, but little is known about its molecular structure. Tumor cells promote the secretion of potent permeability factors that cause leakage of fibrinogen from blood vessels (Dvorak et al. 1992). Extravascular clotting occurs due to procoagulants associated with tumor cells. The resulting fibrinogen/fibrin matrix is constantly remodeled during tumor growth as a consequence of fibrinolysis induced by tumor cell-derived plasminogen activators. It is assumed that fibrin/fibrinogen degradation products play a role during escape of metastatic tumor cells from the primary tumor. There are indications that integrin .alpha..sub.v .beta..sub.3, which is known to interact with the RGDS site in the C-terminal region of the .alpha. chain, may be an important tumor cell surface receptor since it is preferentially expressed on invasive melanoma (Felding-Habermann et al. 1992).
From the foregoing discussion, it becomes clear that significant gaps exist in the understanding of the thrombotic process. Intelligent management of patients depends upon accurate and precise understanding of their thrombotic states. The present state of the art lacks the probes and methods necessary to enable such determinations.
As a result, there exists a need for highly specific, sensitive and reproducible probes for enhancing the understanding of the structure and function of fibrinogen, especially in relation to the generation and function of fibrinopeptides A and B by thrombin cleavage. Accordingly, there exists a need for probes suitable for the specific detection and purification of fibrinopeptide B and fibrinogen incorporating the peptide. In addition, means for diagnostic testing of subjects with respect to the amount and distribution of fibrinogen in the body, as well as monitoring of the generation of fibrinopeptide B as an index of in vivo thrombin activity (i.e., thrombotic state), are needed. The present invention effectively addresses these and other needs for the first time.